【作者】 潘闻燕；

【导师】 朱小东；

【作者基本信息】 广西医科大学 ， 肿瘤学， 2012， 硕士

【摘要】 研究背景及目的：鼻咽癌是我国南方地区的高发肿瘤,其特殊的临床解剖部位和生物学行为,使得放疗被认为是首选的治疗手段。近年来,随着计算机技术的进步和加速器设备的升级,使得三维适形放射治疗(3-dimensional conformal radiotherapy,3-DCRT)技术、调强适形放射治疗(intensity modulated radiotherapy, IMRT)技术得以实现。3-DCRT技术可以实现放射剂量分布在空间三维方向上与肿瘤形状一致,而IMRT不仅剂量分布与肿瘤形状一致,而且剂量强度分布也可以调节,这使得鼻咽癌的5年生存率大大提高,尤其是早期患者的5年生存率可达80%-90%。然而,临床上仍有部分病人尤其是中晚期患者放疗后出现局部残留或局部复发,其中肿瘤细胞在放疗过程中的放射抗拒性是重要原因之一。自噬(autophagy)是一个古老的生物学现象,最早来源于希腊语,其字面意思是自我吞噬,广泛存在于酵母和其它较低级生命体中,是生物降解胞内蛋白、完成细胞器转化、保持内环境稳定的重要方式,也是哺乳动物清除癌细胞的手段之一。近年来的研究显示：自噬是不同于凋亡的另一种程序性细胞死亡,是长寿命蛋白和细胞器的代谢主要依靠途径之一。在人类自噬过程中比较明确的和研究较多的是Beclin1基因和其编码蛋白。自噬体形成必需的微管相关蛋白轻链3(MAPLC3)和p62蛋白也常常用于自噬的检测。为了维持机体细胞内环境的稳态,正常情况下细胞的自噬水平维持在一个较低的水平,当细胞受到一些外界刺激时便可以迅速上调,如：营养缺乏、生长激素缺乏、细胞结构重建、细胞内出现过多的受损细胞器或代谢废物等。自噬过程主要受两种营养感受器的调节：即mTOR(Mammalian Target of Rapamycin)途径依赖的自噬和不依赖mTOR途径的自噬。TOR激酶(target of rapamycin(TOR)kinase)是氨基酸、ATP和激素的感受器,对细胞的分化生长具有重要调节作用,抑制自噬的发生,发挥“门卫(gate-keeper)"作用,在营养充分时关闭自噬信号。而真核起始因子2激酶Gcn2(eukaryoticinitiation factor2(eIF2)kinase Gcn2)及其下游靶位Gcn4——即自噬基因的转录反式作用子,在细胞营养缺乏时则启动自噬信号。位于TOR激酶下游的主要是一些编码自噬ATG蛋白的相关基因,参与自噬的发生、融合、成熟、再循环。雷帕霉素及其类似物是TOR受体的拮抗剂,因此能够诱导自噬。另外,较常用的自噬抑制剂有氯喹(Chloroquine disphosphate)、二磷酸氯喹及3-甲基腺嘌呤(3MA)等。自噬在肿瘤细胞的发生、发展和治疗敏感性中具有双向作用。在肿瘤细胞形成早期刺激自噬能够清除受损细胞器,分解细胞内过多的有害物质(如受损的线粒体等),可以阻止正常细胞向肿瘤细胞的转化。然而,肿瘤细胞中晚期,刺激自噬可清除因受电离辐射、化疗药物等细胞毒性刺激后的受损线粒体和大分子物质,达到营养物质的再循环利用,阻断线粒体的凋亡信号级联传导,使肿瘤细胞逃避凋亡。另外,在肿瘤治疗中,放疗或者化疗联合调节自噬表达可以协同或拮抗肿瘤细胞生长,具体表现在肿瘤放化疗中抑制或诱导自噬可以改变肿瘤细胞的凋亡率。因此,在放疗或者化疗中通过抑制自噬导致肿瘤细胞无法清除受损的细胞器和大分子物质,从而促进肿瘤细胞死亡,增强肿瘤的治疗效果。为了明确自噬和鼻咽癌的关系,以及调节自噬的表达后可能对鼻咽癌产生的放疗增敏效果,本研究采用细胞和分子生物学手段,选取人鼻咽低分化鳞癌细胞株CNE-2细胞为对象,观察了CNE-2细胞在经不同剂量X线照射后自噬和凋亡水平,并且检测了CNE-2细胞受照射及照射联合自噬诱导剂、抑制剂处理后自噬相关蛋白的表达状态和细胞凋亡率。研究了改变人鼻咽癌细胞系CNE-2细胞的自噬活性后,CNE-2细胞对于放射治疗敏感性的变化。探明自噬对照射所致CNE-2细胞凋亡的保护性作用,为提高鼻咽癌放疗敏感性提供实验依据。研究方法：1、先利用蛋白印迹(Western-blot)技术检测CNE-2细胞经不同浓度二磷酸氯喹(Chloroquine disphosphate, CDP)和雷帕霉素(rapamycin)处理后自噬标记蛋白微管相关蛋白轻链3(LC3-Ⅱ)和p62蛋白的表达情况。再通过流式细胞术(Flow cytometry, FCM)技术检测CNE-2细胞分别经抑制自噬最明显的二磷酸氯喹(CDP)浓度、诱导自噬最显著的雷帕霉素(rapamycin)浓度处理后细胞的凋亡率。得到一个抑制自噬和诱导自噬最显著而不影响细胞增殖、凋亡的药物浓度,为后续实验奠定基础。2、利用蛋白印迹(Western-blot)技术检测CNE-2细胞经不同剂量(0、2、4、6、8、10Gy)6Mv-X线照射后不同时间(24h、48h)CNE-2细胞自噬的情况,确定照射是否激活CNE-2细胞自噬,观察自噬与照射剂量和照射后时间的相关性,确定一个诱导自噬最显著的照射剂量和照射后时间点,为下一步实验奠定基础。同时采用流式细胞术(FCM)检测CNE-2细胞经不同剂量(0、2、4、6、8、10Gy)6Mv-X线照射后不同时间(24h、48h)CNE-2细胞的凋亡率,分析照射剂量及照射时间与凋亡的相关性,从而进一步分析自噬与凋亡之间的内在联系。最后继续利用蛋白印迹(Western-blot)技术和流式细胞术(FCM)分别检测CNE-2细胞经单纯10Gy照射、单纯自噬抑制剂二磷酸氯喹(Chloroquine disphosphate, CDP)、单纯自噬诱导剂雷帕霉素(rapamycin)、照射(10Gy)联合二磷酸氯喹(CDP)、照射(10Gy)联合雷帕霉素(rapamycin)及空白对照组6种不同条件处理后自噬标记蛋白微管相关蛋白轻链3(LC3-Ⅱ)和P62蛋白的表达水平变化及细胞存活和凋亡情况。探明自噬在照射所致CNE-2细胞凋亡中的促存活作用。研究结果：1、Western-blot结果显示CNE-2细胞经不同浓度二磷酸氯喹(CDP)、和雷帕霉素(rapamycin)处理后微管相关蛋白轻链3(LC3-Ⅱ)和P62蛋白均不同程度表达。CDP处理组中40μM浓度组LC3-Ⅱ和p62表达水平均明显高于其他各浓度组(F=719.388, F=73.885,P<0.01); rapamycin处理组中20nM浓度组LC3-Ⅱ蛋白表达水平明显高于其他各浓度组(F=375.12,P<0.01),而p62蛋白表达水平却明显低于其他浓度组(F=70.770, P<0.01)。FCM结果示40u M浓度CDP处理组及20nM浓度rapamycin处理组间细胞凋亡率与对照组间无明显差异(F=0.371,P=0.705)。2、 western-blot技术显示随着照射剂量的增加(0、2、4、6、8、10Gy)及照射后时间的延长(24h、48h), LC3-Ⅱ蛋白表达水平呈递增趋势(F=35.84,P<0.01),P62蛋白表达水平呈递减趋势(F=52.47,P<0.01),当10Gy照射后48h时LC3-Ⅰ全部转化成LC3-Ⅱ, p62表达水平最低,自噬现象最明显。单纯CDP组、单纯rapamycin组、单纯10Gy照射组、照射联合CDP组及照射联合rapamycin组LC3-Ⅱ蛋白表达水平较对照组均上调,其中照射联合rapamycin组LC3-Ⅱ蛋白表达水平明显高于其他各组(F=128.03,P<0.01),而该组P62蛋白表达水平显著低于其他5组(即单纯CDP组、单纯rapamycin组、单纯10Gy照射组、照射联合CDP组)(F=117.52, P<0.01); rapamycin组P62表达水平下调,CDP组P62表达水平无明显变化；照射联合CDP组P62蛋白表达水平较其他5组(即单纯CDP组、单纯rapamycin组、单纯10Gy照射组、照射联合rapamycin组)显著上调(F=428.70,P<0.01)。流式细胞术(FCM)结果显示随着照射剂量的增加(0、2、4、6Gy)及照射后时间的延长(24h、48h)CNE-2细胞凋亡率呈现递增趋势,但当单次剂量达8、10Gy时凋亡率递增趋势趋于平坦,细胞凋亡率不再是单纯的剂量递增趋势。后续实验中照射联合CDP组细胞凋亡率显著高于其他5组(F=231.68,P<0.01)；而照射联合rapamycin组细胞凋亡率低于单纯10Gy照射组(F=79.175,P<0.01)。单纯照射组、单纯CDP处理组及单纯rapamycin处理组三组间细胞凋亡率差异无统计学意义(t=-1.198; t=-3.222; t=-1.902.p均>0.05)研究结论：1、二磷酸氯喹(CDP)可以抑制CNE-2细胞的自噬功能,且当在40μ M浓度时抑制功能最显著,而不引起细胞凋亡；雷帕霉素(rapamycin)可以诱导CNE-2细胞发生自噬,且在20nM浓度时诱导自噬现象最明显,而不影响细胞增殖。2、单纯照射可以诱导CNE-2细胞发生自噬和凋亡,其中自噬与照射剂量和照射时间呈现一定量效和时效关系,单次剂量10Gy照射后48h时CNE-2细胞自噬水平最高；在单次照射剂量≤6Gy时细胞凋亡率亦呈现一定量效和时效关系,单次照射剂量8Gy、10Gy照射后48h凋亡率基本达一个平台期。3、抑制自噬可以促进照射所致的人鼻咽癌低分化鳞癌CNE-2细胞凋亡。反之,诱导自噬抑制可以抑制照射所致CNE-2细胞的凋亡,故在人鼻咽癌细胞中,自噬对照射引起的细胞凋亡有一定的保护作用。抑制自噬可能会提高鼻咽癌患者放疗疗效。更多还原

【Abstract】 Background and objectives:Nasophryngeal carcinoma (NPC) is one of the common malignant tumors in China, especially in the southern part of the country. Radiotherapy is considered to be a prevalent treatment in present. In recent years, with advances in computer technology and the upgrade of the accelerator equipment、 through the three-dimensional conformal radiation therapy (3-dimensional conformal radiotherapy,3-DCRT) technology and toneconformal radiation therapy (intensity modulated radiotherapy, IMRT) are come true. The3-DCRT technology is characterized by distributing the radiation dose in the three dimensions of the space consistented with the tumor shape. But the IMRT is a kind of new technology which is complying distribution consistented with tumor shape while the dose intensity distribution can also be adjusted. For this, the survival rate of nasopharyngeal carcinoma was significantly improved, especially the five-year survival rate of patients with early staying up to80%-90%. However, some clinical patients present local recurrence and local residue after radiotherapy, one of the important reasons is due to the radiation resistance in cancer cells. Autophagy, or "self-eating," is a very ancient phenomena in the course of life evolution, which existed widely in yeast and other lower creatures, is an important way in which protein degradated and organelles eliminated to keep cell homeostatic, it is also a process to get rid of cancer cells in mammals. It was first described as an important cellular process almost50years ago in Greece. Datas from recent years indicated that autophagy is considered to be another Programmed cell death path to distinguish from cell apoptosis, represents a dynamic lysosomal pathway responsible for degrading organelles and long-lived proteins. Autophagy is a very primitive biological phenomena in the normal cells physiological processes. Many micromolecule protein participated in autophagy, what we understood well is Beclinl gene and Beclinl protein, microtubule-associated protein light chain3(MAPLC3) and p62is another mark for autophagy some immunodetection..The basal autophagy level of cells keep lowly to maintain homeostatic situationin usually, autophagy level is up-regulated rapidly when crisis come. For example, Nutritional deficiencies^growth factor deficiency、 cell reconstruction or too much damaged cell organelle or metabolic waste.The main regulater of nutrition sensor regulates autophagy is:target of rapamycin(TOR)kinase and eukaryotic initiation factor2(eIF2)kinase Gcn2. TOR play an important role as a gate keeper in cell growth and inhibite autophagy occur. TOR shut off autophagy signal when nutrition adequacy. While eIF2start autophagy signal when starvation.There are many genes lies down-stream of TOR kinase,which participate autophagy some development、 confluence、 maturement、recycling. Rapamycin and the analogue is antagon for TOR receptor and induced autophagy, while Chloroquine disphosphate (CDP) and3-methyladenine (3MA) were usually used to inhibit autophagy formation. Autophagy level is closely related cancer cells occurrence, development and sensitivity of cancer therepy. At early stage of cancer cell formation, provoked autophagy help toeliminate damaged cell organelle^and degrade the noxious substance in endochylema (eg:damaged mitochondria) as well, pretect normal cells from cancerization. However, in late stage of tumor development, autophagy offers precursors such as amino acids, fatty acids and nucleotides to be recycled and used for macromolecule synthesis. It can also block the mitochondrial apoptosis signaling cascade spread, eventually lead to the tumor cells to evade apoptosis. It can be activated as adaptive response to adverse environmental conditions, such as chemotherapy and radiotherapy, down-regulation of autophagy level would make organelles elimination breakdown and accelerate cell death to improve therapeutic efficacy.In order to investigate the relationship between autophagy and NPC and the possibility sensitization effect of radiotherapy after autophagy expression alteration, the cellular and molecular biology methods were used to detected the autophagy-related protein expression level of CNE-2cells to elucidate the relationship of autophagy level and irradiation; investigated changes of radiotherapy sensitivity and autophagy correlated protein when autophagy level of CNE-2altered. We also detect the CNE-2cells apoptosis rate to explain its mechanism to explain the association between autophagy and apoptosis. To investigate the protective function of autophagy in the CNE-2cells apoptosis induced by irradiation.Methods1. Firstly, western-blot were used to detect the expression level of autophagy-related protein LC3-Ⅱ(microtubule-associated protein1light chain3,MAP1-LC3-Ⅱ) and p62in nasopharyngeal carcinoma CNE-2cells, which were under different concentration Chloroquine disphosphate(CDP) and different concentration rapamycin treated. Then, Flow cytometry (FCM) was used to detect the ration of apoptosis of CNE-2cells, which were treated with three different conditions.2. Western-blot and Flow cytometry (FCM) were used again to detect autophagy and apoptosis in nasopharyngeal carcinoma CNE-2cells which were under different doses (0,2,4,6,8and lOGy) X-ray radiation at different time (24h and48h) to determine the relationship between cell autophagy and apoptosis after irradiation. Then, Western-blot and FCM were used again to detect the level of autophagy-related protein LC3-Ⅱ and p62expression and the ration of apoptosis of CNE-2cells, which were treated with six different conditions as irradiation only(10Gy), Chloroquine disphosphate(CDP) treated only, rapamycin treated only, Chloroquine disphosphate combine with lOGy irradiation, rapamycin combine with10Gy irradiation and the blank control group. Which were in order to define the protection function of autophagy in CNE-2cells.Results1.when CNE-2cells was treated with40P M Chloroquine disphosphate (CDP), the expression level of LC3-Ⅱ and p62protein both increased more pronounced than other CDP groups (P<0.01). In rapamycin treated groups, the level of LC3-II protein expression was significantly increased after20nM rapamycin treated, but the level of p62expression decreased, there were significant differences between various groups (P<0.01). And the ration of apoptosis is no difference among control group,40y M CDP group and20nM rapamycin group (P>0.01).2. With the increase of irradiation doses (0,2,4,6,8and lOGy) and the elongation of irradiation time (24and48h), the phenomenon of autophagy increased significantly. While the apoptosis rate of CNE-2cells were increased with irradiation dose at48h post-irradiation, there were significant differences between various groups(P<0.05), Compared with irradiation alone, Chloroquine disphosphate(CDP) combined with irradiation significantly increased cell apoptosis, as well as p62and LC3-II protein level. However, rapamycin combined with irradiation induced strong upregulation of LC3-II expression but decreased of cell apoptosis and p62level. The ration of cells apoptosis among only(10Gy) irradiation, Chloroquine disphosphate(CDP) treated only and rapamycin treated only groups is no significant differences (t=-1.198; t=-3.222; t=-1.902, p>0.05).Conclusion Firstly, Chloroquine disphosphate can inhibit autophagy in CNE-2cells? the inhibiton is conspicuous at40μM concentration. While rapamycin can induce autophagy and the induction is extraordinary at20nM concentration. Otherwise, at the two conditions, it will not impact the ration of apoptosis of CNE-2cells. Secendly, irradiation can induce autophagy and apotosis in CNE-2cells. Autophagy level is increased depend on the radiation dose and exposure time. When the single dose is less then or eaqual6Gy, the cells apoptosis ration is also increased belong dose and time. Third, inhibition of autophagy increased irradiation-induced apoptosis, while induction of autophagy protected cell survial. It suggests that autophagy might play a role as a self-defense mechanism in cancer radiotherapy.更多还原